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LastUpdated:9/7/16
TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab
Tutorial:HowtousetheWheatTILLINGdatabase
1. Visithttp://dubcovskylab.ucdavis.edu/wheat_blasttogototheBLASTpageorclickonthe‘WheatBLAST’buttononthehomepage.
2. A)Pasteinyournucleotidesequence,B)selectthe‘TILLINGGenomicReference(4xand6x)’database,andthenC)hitthe“Basicsearch”button.
3. Wait5-20secondswhiletheBLASTsearchruns.Thetimeisdependentonnumberofquerysequencesandthelengthofthequery.
A)
B)
C)
LastUpdated:9/7/16
TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab
4. Whenfinished,youwillbepresentedwithatableofBLASThitresults.Here,A)youcanviewtheBLASTHTMLoutput,B)downloadtabularBLASTTSVoutput,C)checktheboxforcontigsequenceyouwanttodownload,D)clickonScorestoviewtheBLASTHTMLalignmentforaspecifichit,andE)clickalinktoviewtheannotationwithpossiblemutantlinesinthesameregion.Fromthispage,youcanalsodownloadfullcontigsequencesbasedonyourresults.
5. Afterclickingonavisualizationlink,allowJBrowsetoloadthesequenceandannotationdata.Thismaytakeanywherefrom5secondsto3minutesdependingonconnectionspeed.Oncefinishedloading,youwillhaveyourhitregionhighlightedinyellow,withyourBLASTHSPsdepictedbygreenbarsintheBlastHSPResultstrack.BelowtheBlastHSPResultstrack,wecanseetheHighConfidenceMutantstrackwhereSingleNucleotideVariants(SNVs)arecolor-codeddependingontheseverityofthevarianteffect.SNVsthataremostseverearecoloredinRED(stop_gained,stop_lost,splice_sitechanges),mediumseveritySNVsarecoloredinPURPLE(missense/aminoacidchange),theleastsevereSNVsarecoloredinGREEN(synonymous/noaminoacidchange),andlastlySNVsarecoloredinBLUEforintron_variantsorwhentheeffectisunknown(I.E.inthecaseofallUCWchromosomeassembledcontigsoranythingthatisnotannotatedinIWGSC).
A)
B)
C) D) E)
LastUpdated:9/7/16
TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab
6. WhenclickingonaparticularSNV,aninformationwindowwillbedisplayedwithdetails.
7. Inordertodownloadnucleotidesequencedataforonlyaparticularregion,youcanusetheReferenceSequencetracktoexportthesequenceinFASTAformat.First,zoomintotheregionofinterestsothatitisdisplayedinthevisualizationwindow.Tozoom,holdthe<SHIFT>keyanduseyourmousetoclickanddragovertheexactregionyouwant.YoushouldseeaREDlinewiththepositionalstartinformationafterpressingSHIFT.
LastUpdated:9/7/16
TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab
8. Then,clickonthereferencetrackdownarrowandchoose‘Savetrackdata’.Thistrackmaybeatthebottomoftheinterface.
9. Youwillthenbepresentedwithadialogwindowwhereyoucanchoosefromacoupleofoptions:Highlightedregion,visibleregion,orwholereferencesequence.Inthiscase,sincewearetryingtograbonlyaparticularsub-regionofthehighlightedregion,wewillchoose‘Visibleregion’andthenclickthe‘Save’button
10. TodownloadSNPdatainTSVformat(canbeopenedinexcel),right-clickonaSNPfordifferentexportoptions.
LastUpdated:9/7/16
TutorialbyHansVasquez-GrossUCDavis-DubcovskyLab
Alternatedownloadoption:
• DownloadingmutationdatacanalsobesearchedbasedonIDsfromtheSampleSearchpage:http://dubcovskylab.ucdavis.edu/wheat-tilling/sample-search
• Thefirst3options(LineName,GeneName,orScaffoldName)willsearchautomaticallyandpresentyouwithalistofoptions.Clickingononeinthedropdownboxwillautomaticallydownloadthemutationdataforthesearchterm.PleasenotethesearchisCASESENSITVE.
• Additionally,thebottomboxcanbeusedtopasteinmixedIDlisttoautomaticallygenerateonefileforallthesearchedmutationdata.